The objective of this research plan is to test the hypothesis that three conserved protein antigens from Streptococcus pneumoniae can induce protective responses when delivered via a live attenuated S. Typhi vector strain. The use of protein antigens conserved on all pneumococcal serotypes as vaccine components has attracted a great deal of attention as a promising alternative to capsular polysaccharide-based vaccines do to the potential of inducing broad spectrum immunity against diverse pneumoccal serotypes. Three conserved pneumococcal proteins, including PsaA, PspA, and Ply have been shown to be immunogenic and to elicit protective responses in animal models; specifically, PsaA and PspA have been shown to reduce pneumococcal carriage while Ply and PspA protect against invasive challenge. Here we will test a novel delivery method by using live, oral, attenuated S. Typhi vaccine strains as delivery vectors expressing combinations of these critical antigens in a single formulation with which it might be possible to engender responses protective against both carriage and invasive phases of pneumococcal disease. In order to accomplish this, the three pneumococcal antigens must be expressed in optimal immunogenic conformation in the S. Typhi vector strain. Aim 1 will address optimization of expression of each individual antigen in S. Typhi by engineering plasmids directing protein expression in the optimal bacterial compartment, utilizing preferred promoters and expressing immunogenic fragments comprising protective epitopes. Aim 2 will utilize the optimal constructions of each individual antigen in engineering multi-antigen plasmids optimized for expression of all components. Aim 3 will evaluate the functional capacity of immune responses generated in mice immunized with S. Typhi derivatives expressing the pneumococcal antigens by utilizing two challenge models, one testing reduction in pneumococcal carriage and the second evaluating protection from lethal invasive challenge with wild type S. pneumoniae. A successful live vector-based protein antigen pneumococcal vaccine offers advantages as a needle free, inexpensive formulation capable of protecting against a broad range of pneumococcal serotypes. These studies will contribute knowledge of the protective capabilities of three pneumococcal protein candidate antigens individually and in mixtures, extend the use of S. Typhi live vector strains, and provide preclinical data necessary for advancing promising candidates into clinical trials.